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采用以β-actin基因为内参基因的实时荧光定量PCR方法,检测了不同大小近江牡蛎(Crassostrea hongkongensis)被100μg/L Cu2+处理48 h后闭壳肌、鳃、外套膜、消化腺等四种组织器官中HSC70基因mRNA的表达水平。结果显示,大个体的外套膜、鳃和消化腺中HSC70基因mRNA表达量均明显高于小个体,而在闭壳肌中大小个体表达量无显著差异。同时用不同浓度的Cu2+(0.01、0.1、1,10、50、100μg/L)处理个体大小相似的近江牡蛎不同时间(6 h,2 d,4 d,8 d,20 d,35 d和45 d)后,检测牡蛎外套膜、鳃和消化腺中HSC70基因的表达水平,结果显示用Cu2+连续浸泡处理时,近江牡蛎外套膜和鳃中可诱导HSC70基因显著高表达的Cu2+临界浓度在1~10μg/L范围内,消化腺中可诱导HSC70基因显著高表达的Cu2+临界浓度在0.1~1μg/L范围内。在可诱导HSC70基因表达的浓度组中,在鳃和消化腺中显著高表达峰值出现在第2 d,到第8 d恢复至对照水平;而外套膜中表达峰值出现在第4 d,但延续时间最长可至第20 d。由此可见,近江牡蛎HSC70基因对Cu2+的刺激反应非常灵敏,且持续时间较长,可作为Cu2+污染的早期预警分子。
Using real-time PCR with β-actin as a reference gene, four tissues of the muscle, gill, mantle and digestive gland of Crassostrea hongkongensis were treated with 100 μg / L Cu2 + for 48 h The level of HSC70 gene mRNA expression in organs. The results showed that the mRNA expression of HSC70 in mantle, gill and digestive gland of large individuals was significantly higher than that in small individuals, while there was no significant difference in the expression of HSC70 in large individuals. At different time (6 h, 2 d, 4 d, 8 d, 20 d, 35 d and 45 h) with different concentrations of Cu2 + (0.01,0.1,1,10,50 and 100μg / L) d), the expression level of HSC70 gene in mantle, gill and digestive gland of oyster was detected. The results showed that the critical concentration of Cu2 +, which can induce HSC70 gene in mantle and gill of oyster oyster, In the range of 10μg / L, the critical concentration of Cu2 +, which can induce HSC70 gene expression in the digestive gland, is in the range of 0.1 ~ 1μg / L. In the concentration-inducing HSC70 group, the peak of high expression in the gill and digestive gland appeared on the 2nd day and returned to the control level on the 8th day, while the peak appeared on the 4th day in the mantle but continued Up to the 20th day. Thus, HSC70 gene of Oryx oyster is very sensitive to Cu2 + stimulation and lasts for a long time and can be used as an early warning molecule for Cu2 + pollution.